Protein overexpression based on introduction of multiple gene copies is well established. To improve purification or quantification, proteins are typically fused to peptide tags. In Saccharomyces ...cerevisiae, this has been hampered by multicopy toxicity of the TAP and GFP cassettes used in the global strain collections. Here, we show that this effect is due to the EF‐1α promoter in the HIS3MX marker cassette rather than the tags per se. This promoter is frequently used in heterologous marker cassettes, including HIS3MX, KanMX, NatMX, PatMX and HphMX. Toxicity could be eliminated by promoter replacement or exclusion of the marker cassette. To our knowledge, this is the first report of toxicity caused by introduction of a heterologous promoter alone.
► We examine the reported toxicity of TAP tagging proteins. ► TAP tagging does not generally cause loss of protein function. ► The MX marker cassettes are toxic to yeast at high copy numbers. ► Toxicity is due to the high copy presence of the heterologous TEF1 promoter. ► Genomic single copy integration of the MX marker and TEF1 promoter is not toxic.
Previously, we found that amplification of chromosome 17q24.1-24.2 is associated with lymph node metastasis, tumour size, and lymphovascular invasion in invasive ductal carcinoma. A gene within this ...amplicon, CACNG4, an L-type voltage-gated calcium channel gamma subunit, is elevated in breast cancers with poor prognosis. Calcium homeostasis is achieved by maintaining low intracellular calcium levels. Altering calcium influx/efflux mechanisms allows tumour cells to maintain homeostasis despite high serum calcium levels often associated with advanced cancer (hypercalcemia) and aberrant calcium signaling.
In vitro 2-D and 3-D assays, and intracellular calcium influx assays were utilized to measure tumourigenic activity in response to altered CANCG4 levels and calcium channel blockers. A chick-CAM model and mouse model for metastasis confirmed these results in vivo.
CACNG4 alters cell motility in vitro, induces malignant transformation in 3-dimensional culture, and increases lung-specific metastasis in vivo. CACNG4 functions by closing the channel pore, inhibiting calcium influx, and altering calcium signaling events involving key survival and metastatic pathway genes (AKT2, HDAC3, RASA1 and PKCζ).
CACNG4 may promote homeostasis, thus increasing the survival and metastatic ability of tumour cells in breast cancer. Our findings suggest an underlying pathway for tumour growth and dissemination regulated by CACNG4 that is significant with respect to developing treatments that target these channels in tumours with aberrant calcium signaling.
Canadian Breast Cancer Foundation, Ontario; Canadian Institutes of Health Research.
The combination of immune checkpoint blockade with chemotherapy is currently under investigation as a promising strategy for the treatment of triple negative breast cancer (TNBC). Tumor-associated ...macrophages (TAMs) are the most prominent component of the breast cancer microenvironment because they influence tumor progression and the response to therapies. Here we show that macrophages acquire an immunosuppressive phenotype and increase the expression of programmed death ligand-1 (PD-L1) when treated with reactive oxygen species (ROS) inducers such as the glutathione synthesis inhibitor, buthionine sulphoximine (BSO), and paclitaxel. Mechanistically, these agents cause accumulation of ROS that in turn activate NF-κB signaling to promote PD-L1 transcription and the release of immunosuppressive chemokines. Systemic in vivo administration of paclitaxel promotes PD-L1 accumulation on the surface of TAMS in a mouse model of TNBC, consistent with in vitro results. Combinatorial treatment with paclitaxel and an anti-mouse PD-L1 blocking antibody significantly improved the therapeutic efficacy of paclitaxel by reducing tumor burden and increasing the number of tumor-associated cytotoxic T cells. Our results provide a strong rationale for the use of anti–PD-L1 blockade in the treatment of TNBC patients. Furthermore, interrogation of chemotherapy-induced PD-L1 expression in TAMs is warranted to define appropriate patient selection in the use of PD-L1 blockade.
Cancer cells have higher reactive oxygen species (ROS) than normal cells, due to genetic and metabolic alterations. An emerging scenario is that cancer cells increase ROS to activate protumorigenic ...signaling while activating antioxidant pathways to maintain redox homeostasis. Here we show that, in basal-like and BRCA1-related breast cancer (BC), ROS levels correlate with the expression and activity of the transcription factor aryl hydrocarbon receptor (AhR). Mechanistically, ROS triggers AhR nuclear accumulation and activation to promote the transcription of both antioxidant enzymes and the epidermal growth factor receptor (EGFR) ligand, amphiregulin (AREG). In a mouse model of BRCA1-related BC, cancer-associated AhR and AREG control tumor growth and production of chemokines to attract monocytes and activate proangiogenic function of macrophages in the tumor microenvironment. Interestingly, the expression of these chemokines as well as infiltration of monocyte-lineage cells (monocyte and macrophages) positively correlated with ROS levels in basal-like BC. These data support the existence of a coordinated link between cancer-intrinsic ROS regulation and the features of tumor microenvironment. Therapeutically, chemical inhibition of AhR activity sensitizes human BC models to Erlotinib, a selective EGFR tyrosine kinase inhibitor, suggesting a promising combinatorial anticancer effect of AhR and EGFR pathway inhibition. Thus, AhR represents an attractive target to inhibit redox homeostasis and modulate the tumor promoting microenvironment of basal-like and BRCA1-associated BC.
► We examine the reported toxicity of TAP tagging proteins. ► TAP tagging does not generally cause loss of protein function. ► The MX marker cassettes are toxic to yeast at high copy numbers. ► ...Toxicity is due to the high copy presence of the heterologous TEF1 promoter. ► Genomic single copy integration of the MX marker and TEF1 promoter is not toxic.
Protein overexpression based on introduction of multiple gene copies is well established. To improve purification or quantification, proteins are typically fused to peptide tags. In Saccharomyces cerevisiae, this has been hampered by multicopy toxicity of the TAP and GFP cassettes used in the global strain collections. Here, we show that this effect is due to the EF-1α promoter in the HIS3MX marker cassette rather than the tags per se. This promoter is frequently used in heterologous marker cassettes, including HIS3MX, KanMX, NatMX, PatMX and HphMX. Toxicity could be eliminated by promoter replacement or exclusion of the marker cassette. To our knowledge, this is the first report of toxicity caused by introduction of a heterologous promoter alone.
PDF
